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. 2017 Aug 10;8(41):69237–69249. doi: 10.18632/oncotarget.20144

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Copyright: © 2017 Tagde et al.

This is an open-access article distributed under the terms of the Creative Commons Attribution License 3.0 (CC BY 3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

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A. RPMI8226 cells treated with JQ1 or vehicle control for 48 h were analyzed for RING2 mRNA levels by qRT-PCR (left). The results (mean±SD of 3 determinations) are expressed as relative mRNA levels as compared with that obtained for the vehicle treated cells (assigned a value of 1, left). Lysates were immunoblotted with the indicated antibodies (right). B. RPMI8226/Tet-CshRNA and RPMI8226/Tet-MYCshRNA cells treated with DOX for 5 d were analyzed for RING2 mRNA levels by qRT-PCR (left). The results (mean±SD of 3 determinations) are expressed as relative mRNA levels as compared with that obtained for the Tet-CshRNA cells (assigned a value of 1). Lysates were immunoblotted with the indicated antibodies (right). C. Schema of the RING2 promoter with highlighting of putative MYC binding sites. Soluble chromatin from the RPMI8226/CshRNA and RPMI8226/MUC1shRNA (left) and OPM-2/CshRNA and OPM-2/MUC1shRNA (right) cells was precipitated with anti-MYC or a control IgG antibody. The final DNA samples were amplified by qPCR with pairs of primers (Supplementary Table S2) encompassing the MYC binding sites in the RING2 promoter. The results (mean±SE of 3 determinations) are expressed as the relative fold enrichment compared with that obtained for the IgG control (assigned a value of 1). D. RPMI8226/CshRNA and RPMI8226/NF-κBshRNA were analyzed for RING1 mRNA levels by qRT-PCR (left). The results (mean±SD of 3 determinations) are expressed as relative mRNA levels as compared with that obtained for the CshRNA cells (assigned a value of 1). Lysates were immunoblotted with the indicated antibodies (right). E. RPMI8226 cells treated with the 5 μM BAY-11-7085 or vehicle control for 24 h were assessed for RING1 levels by qRT-PCR (left). The results (mean±SD of 3 determinations) are expressed as relative mRNA levels as compared with that obtained for the vehicle treated cells (assigned a value of 1). Lysates were immunoblotted with the indicated antibodies (right). F. Schema of the RING1 promoter with highlighting of putative NF-κB p65 binding sites. Soluble chromatin from the RPMI8226/CshRNA and RPMI8226/MUC1shRNA (left) and OPM-2/CshRNA and OPM-2/MUC1shRNA (right) cells was precipitated with anti-NF-κB p65 or a control IgG antibody. The final DNA samples were amplified by qPCR with pairs of primers (Supplementary Table 2) encompassing the NF-κB p65 binding sites in the RING1 promoter. The results (mean±SE of 3 determinations) are expressed as the relative fold enrichment compared with that obtained for the IgG control (assigned a value of 1).